1. Field of the Invention
The present invention is broadly concerned with an improved method for the quantitative determination of glycated proteins in a liquid sample such as blood or other body fluids, extracts or emulsions (e.g., urine) containing both glycated and nonglycated protein fractions. More particularly, it is concerned with such a method which makes use of spectrophotometric quantitation employing balanced mobile phases during the analysis which absorb as background substantially equal quanta of light. In this fashion, the analysis can be conducted using automated equipment and with the complete elimination of the need for blanking of the spectrophotometer. A novel electronic control of the automated analysis is also disclosed.
2. Description of the Prior Art
The measurement of glycated protein in the blood of a patient suffering from diabetes mellitus provides an attending physician with a means for assessing blood sugar control over different periods of time. As a consequence, considerable research has been done in the past to develop accurate, rapid analyses for glycated proteins. In order to be truly effective, however, such methods should also be capable of measuring the glycated protein content of both hemoglobin and plasma (serum) proteins. This stems from the fact that the percentage of glycated hemoglobin in a sample is a measure of mean blood sugar concentrations for the preceding 45 to 60 days, whereas the percentage of glycated plasma protein reflects blood sugar concentrations over a shorter period, approximately 1-3 weeks prior to analysis.
It is known that non-enzymatically glycated proteins such as hemoglobins and circulating plasma or serum proteins differ from nonglycated species by the attachment of sugar moieties to the proteins at various binding sites by means of a ketoamine bond. Glycated proteins thus contain functional groups (sometimes reported as 1,2-cis-diol groups) not found in nonglycated proteins. The presence of such functional groups thus provides a basis for separation of glycated and nonglycated proteins.
One known technique for separating glycated and nonglycated protein fractions is known as boronate affinity chromatography. This analysis relies on the fact that borates or boronates, such as m-aminophenylboronic acid (PBA), can under certain conditions, bond with the functional groups in glycated proteins. The bonds in the resulting complex are reversible in aqueous solution. In the typical chromatographic system, the boronate is immobilized on a support medium in a chromatographic column. A solution containing the substance under analysis, such as hemoglobin or plasma (serum), is applied to the column, wherein the glycated components bond with the boronate. Nonglycated components are washed out of the column with a suitable buffer solution, then eluated from the column and collected separately. Eluation of the glycated components is achieved by means of either: 1) a buffer solution containing a competing polyol, which replaces the protein in the boronate complex, or 2) a buffer or acid solution of low (below neutrality) pH.
A number of secondary factors, such as interactions between boronates and amines or carboxyls, influence the chromatographic separation. Complex molecules such as proteins also engage in hydrophobic, ionic, hydrogen-bond, and charge-transfer interactions. The need to design chromatographic systems which maximize the specific binding of boronates with diol-containing glycated proteins is therefore complicated by the need to also minimize the binding of nonglycated proteins through nonspecific interactions. Many factors such as buffer composition, molarity, ion content, ligand concentration and pH are known to influence the boronate affinity chromatographic separation of glycated and nonglycated proteins.
Glycated and nonglycated proteins separated chromatographically are commonly quantitated spectrophotometrically. For hemoglobin quantitation, the spectrophotometer is set to measure the absorbance of light in or near the "Soret region" (413-415 nanometers), which is the range of peak absorbance for hemoglobin. For plasma (serum) protein quantitation, the spectrophotometer is set to measure the absorbance of light in the 280 nanometer range, at which the proteins show peak absorbance. Of course, other wavelength determinations can also be maintained using known chromagens in the eluates. The buffer solution used to elute the non-glycated components is first placed in the light path of the spectrophotometer. The spectrophotometer is then adjusted so that zero absorbance is displayed on the scale or readout. This procedure, called a "blank", corrects for the background absorbance of the contents of the buffer solution. The next step is to place the non-glycated protein-containing buffer solution in the light path of the spectrophotometer and record the absorbance of light. By virtue of the "blanking" procedure, any increase in the absorbance of light by the protein-containing solution is due entirely to the protein content.
The same procedure is used for the eluate containing the glycated components; that is, the spectrophotometer is first "blanked" with the elution buffer solution in the light path, after which the increase in absorbance of the protein-containing buffer is recorded. Quantitation is then achieved by calculation. The increase in absorbance over the "blank" solutions of the protein-containing solutions is directly proportional to the protein content. The calculation also takes into account the dilution of the proteins by the buffer solutions. The result of this calculation is the expression of the glycated components as a percentage of the whole, e.g., "10% glycated hemoglobin" or "14% glycated plasma proteins."
The above procedure, although somewhat effective in yielding accurate hemoglobin protein analyses, is generally inaccurate or imprecise in serum protein analyses, and moreover is less suited for commercial laboratories because the protocol is very time consuming and costly. Thus, the need to individually collect and subsequently blank each of the eluates presents a significant drawback in the context of multiple analyses in a large laboratory.